1. Field of the Invention
The present invention relates to a method for producing a target substance using a microorganism. More precisely, the present invention provides a means for improving production of L-amino acids, antibiotics, vitamins, growth factors and bioactive substances using a microorganism.
2. Brief Description of the Background Art
Typical methods for producing substances using microorganisms are known, including methods for producing L-amino acids by fermentation. L-amino acids are used not only as seasonings and foodstuffs, but also as components of various nutritional mixtures for medical purposes. Furthermore, they are used as additives for animal feed, reagents in the drug manufacturing and chemical industries, and growth factors for production of L-amino acids such as L-lysine and L-homoserine using a microorganism. Known microorganisms that can produce L-amino acids by fermentation include the coryneform bacteria, Escherichia bacteria, Bacillus bacteria, Serratia bacteria, and so forth.
During production of target substances by fermentation such as described above, most of the raw materials are known to contain saccharides such as blackstrap molasses. Also, in amino acid or nucleic acid fermentation, the culture is performed using a saccharide as a raw material. Although sugarcane contains abundant amounts of starch, it is rare to use it as a raw material. It is more commonly used as a decomposition product, resulting in, for example, monosaccharides or disaccharides. In these methods, a solution of a saccharide-producing enzyme such as amylase is generally used, and thereby polysaccharide starches are decomposed into relatively low molecular weight saccharides such as glucose, maltose, and maltotriose.
During fermentation of Gram-negative enterobacteria such as Escherichia coli (E. coli), the use of a starch decomposition solution can cause problems. For example, E. coli consumes glucose when it is present as the main component, but it suffers from so-called glucose repression, which means that oligosaccharides containing two or more monosaccharides such as maltose are consumed only after monosaccharides are completely consumed. Therefore, if fermentation is terminated when only glucose, present as the main component of the starch decomposition solution, is consumed, oligosaccharides such as maltose are not assimilated but remain. Furthermore, if the intent is to consume oligosaccharides after consumption of glucose, the culture time must be extended, and therefore utility cost and so forth are wasted.
It is known that E. coli and Salmonella typhimurium generally suffer from glucose repression. That is, when glucose is assimilated with other carbon sources such as lactose, maltose and glycerol, glucose is assimilated first and the other carbon sources are assimilated later. Monod et al. discovered that, when lactose and glucose were the carbon sources, two-phase proliferation, i.e., so-called diauxie, was observed (Monod, J., Growth, 11, 223-247, 1947). Through research in molecular biology, the mechanism thereof has become clear. That is, IIAGlc (glucose PTS enzyme II) acts as a phosphate donor for glucose in the phosphate cascade at the time of assimilation in the glucose-phosphoenolpyruvate-sugar phosphotransferase system, i.e., the so-called PTS system, and exists in a dephosphorylated state. The dephosphorylated IIAGlc causes so-called inducer exclusion, in which the dephosphorylated IIAGlc inhibits uptake of the other saccharides (Postma P. W., Lengeler J. W. and Jacobson G. R.: in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt F. C.), pp. 1149-1174, 1996, ASM Press, Washington D.C.).
Uptake of maltose in E. coli suffers from glucose repression caused by the interaction between the dephosphorylated IIAGlc and the MalK protein, which constitutes the uptake system for maltose by non-PTS. That is, when the bacterium is taking up glucose, IIAGlc exists in excessive amounts in the cell and binds to the MalK protein, resulting in inhibition of maltose uptake. Furthermore, a mutant strain which has improved maltose uptake in the presence of a glucose analogue was also obtained, and this mutant strain has a mutation in the malK gene coding for the MalK protein (Dean D. A. et al., Regulation of the Maltose Transport System of Escherichia coli by the Glucose-specific Enzyme III of the Phosphoenolpyruvate-Sugar Phosphotransferase System., J. Biol. Chem., 265 (34), 21005-21010, 1990; Kuhnau, S. et al., The Activities of the Escherichia coli MalK Protein in Maltose Transport and Regulation, and Inducer Exclusion Can Be Separated by Mutations, J. Bacteriol., 173 (7), 2180-2186, 1991).
Furthermore, and also for IIAGlc, a mutant strain that contained a mutant protein which showed reduced binding with lactose permease has been reported (Zeng, G. A. et al., Mutation analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferease system in Escherichia coli, Res. Microbiol., 143, 251-261, 1992). Lactose permease is an uptake enzyme for lactose, which is a non-PTS saccharide.
However, whether the aforementioned mutant strains can assimilate maltose simultaneously in the presence of glucose has not been reported.